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To study the effects of localized secretion of cytokines on tumor progression, the gene for human interleukin 2 (IL-2) was introduced via retroviral vectors into CMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of IL-2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protective immune response against a challenge with a tumorigenic dose of parental CMS-5 cells. Co-injection of IL-2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation even when highly tumorigenic doses of CMS-5 cells were used. Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumor-bearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d). High levels of tumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if IL-2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets. These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.

There is a need for in vivo monitoring of cell engraftment and survival after cardiac cell transplantation therapy. This study assessed the feasibility and usefulness of combined PET and MRI for monitoring cell engraftment and survival after cell transplantation. Methods: Human endothelial progenitor cells (HEPCs), derived from CD341 mononuclear cells of umbilical cord blood, were retrovirally transduced with the sodium iodide symporter (NIS) gene for reporter gene imaging by 124 I-PET and labeled with iron oxides for visualization by MRI. Imaging and histologic analysis were performed on 3 groups of nude rats on days 1, 3, and 7 after intramyocardial injection of 4 million HEPCs. Results: In vitro studies demonstrated stable expression of functional NIS protein and normal viability of HEPCs after transduction. On day 1, after intramyocardial transplantation, iron-and NIS-labeled HEPCs were visualized successfully on MRI as a regional signal void in the healthy myocardium and on PET as 124 I accumulation. The 124 I uptake decreased on day 3 and was undetectable on day 7, and the MRI signal remained unchanged throughout the follow-up period. Histologic analysis with CD31 and CD68 antibodies confirmed the presence of either labeled or nonlabeled control transplanted HEPCs at the site of injection on day 1 but not on day 7, when only iron-loaded macrophages were seen. Furthermore, deoxyuride-59-triphosphate biotin nick end labeling showed extensive apoptosis at the site of transplantation. Conclusion: The combination of MRI and PET allows imaging of localization and survival of transplanted HEPCs together with morphologic information about the heart. Although iron labeling rapidly loses specificity for cell viability because of phagocytosis of iron particles released from dead cells, reporter gene expression provided specific information on the number of surviving cells. This multimodality approach allows complementary analysis of cell localization and viability.

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.Adenoviruses have developed efficient strategies to force infected cells into the S phase of the cell cycle (1). This process involves the adenoviral E1A and E1B proteins, which are the first viral proteins to be expressed after infection, and both are essential for viral replication (2, 3). Replication of adenovirus DNA depends directly on interactions between the host cell replication factors NFI, NFII, and NFIII (4) and the three viral replication proteins encoded by the E2 region. The adenovirus E2 transcription unit consists of the E2A and E2B genes, which encode precursor terminal protein pTP, DNA polymerase, and DBP, a multifunctional DNA-binding protein (5). E2 gene expression is driven from two promoters. At early times of infection, E2 gene transcription is under control of the E2 early promoter. At intermediate stages of infection, E2 gene expression is controlled by the E2 late promoter (6). E2 early promoter activity is regulated by adenovirus E1A protein, which controls the activity of the E2F transcription factor by targeting the tumor suppressor protein pRB (7,8). In contrast, activity of the E2 late promoter is repressed by E1A (9). The E2 late promoter is characterized by the presence of a TATA box, two SP1 recognition sites, and three CCAAT boxes. Two of the inverted CCAAT boxes are located at positions Ϫ72 and Ϫ135 relative to the E2 late cap site in a 157-bp sequence of the of the E2 late promoter, which is sufficient for efficient E2 gene transcription (10, 11).Inverted CCAAT boxes have been identified as sites for Y box proteins, which are highly conserved through evolution from prokaryotes to eukaryotes, and they can function as transcriptional, translational, and developmental regulators (12)(13)(14). In eukaryotes, increased expression of Y box proteins in somatic cells is associated with drug resistance and a malignant phenotype (15), and it was discussed that Y box proteins are involved in activating certain genes that are expressed in the S phase of the cell cycle (16). Recently, it has...

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